Thermosensitive hydrogel coupled with sodium ascorbyl phosphate promotes human umbilical cord-derived mesenchymal stem cell-mediated skin wound healing in mice

Poor survival and restricted function of transplanted stem cells are regarded as limiting their efficacy in wound recovery greatly. Consequently, it is necessary to identify innovative therapeutic strategies to solve these issues. Firstly, the biological effect of PF-127 hydrogel alone and in combination with SAP on the survival, and migration of cultured HUCMSCs was assessed by cell viability, apoptosis, and scratch wound assays. S. aureus and E. coli were used to evaluate the antibacterial activity of PF-127 plus SAP combination. Further, the ability of HUCMSCs-conditioned medium (HUCMSCs-CM) to promote the angiogenesis and migration of human umbilical vein endothelial cells (HUVECs) in vitro was evaluated using tube formation and transwell migration assays. Finally, the HUCMSCs embedded in PF-127 plus SAP scaffold were administered onto mice’s excisional cutaneous wound bed. Histological and immunohistochemical analyses were employed to investigate the wound healing capacity as well as cellular responses of PF-127/HUCMSCs/SAP hydrogel. PF-127 showed cytotoxicity on HUCMSCs, whereas the addition of SAP significantly promoted cell viability and alleviated apoptosis of HUCMSCs encapsulated in PF-127 hydrogel in vitro. SAP supplementation substantially abrogated the inhibiting effect of PF-127 on the migration of HUCMSCs in vitro. The combination of PF-127 and SAP exerted an obvious bacteriostatic function on S. aureus and E. coli. Moreover, the co-treatment with SAP could remarkably enhance the stimulative effect of HUCMSCs-CM on the angiogenesis and migration of HUVECs in vitro. PF-127 combined SAP-embedded HUCMSCs transplantation resulted in a potently accelerated wound healing process, promoted the number of proliferating cells and newly formed blood vessels, as well as enhanced expression of vascular endothelial growth factor. PF-127 coupled with SAP contributes to HUCMSCs-mediated traumatic wound closure in mice by promoting cell survival, antibacterial action, and angiogenesis. Our results offered a theoretical foundation for the clinical treatment of traumatic skin defects.


Scientific Reports
| (2023) 13:11909 | https://doi.org/10.1038/s41598-023-38666-w www.nature.com/scientificreports/ Cell apoptosis assay. The cell apoptosis was determined in HUCMSCs using the DAPI staining method (BioVision, USA). 5 × 10 4 HUCMSCs with different encapsulation conditions were cultivated in a 48-well plate for 24 h. HUCMSCs were fixed and permeabilized, after which it was dyed with DAPI for 10 min. The results were then observed and captured using an inverted fluorescence microscope (DCM8, Leica, Germany). Randomly select 20 fields of view, calculate the proportion of apoptotic cells, and calculate the average value.
Cell wound scratch assay. 1 × 10 5 HUCMSCs with different encapsulation conditions were cultivated in 6-well plates and incubated in an FBS-free medium for 24 h. Then, a 10 μL pipette tip was employed to scratch a straight line on the cell monolayer. HUCMSCs were imaged at the indicated time point, and cell migration rate was calculated by Image J software. Data has been reported as the extent of wound closure by the initial scratch width.
Tube formation assay. HUVECs were incubated in serum-free MEM-alpha medium with or without HUCMSCs-CM for 12 h. 100 μL of Matrigel (BD Biosciences) was diluted with MEM-alpha medium and coated in 6-well plates at 37 °C for 1 h. Then, 5 × 10 4 HUVECs were seeded on Matrigel. The 6-well plates were transferred back to the incubator overnight. The tube formation ability of HUVECs was photographed and quantified under phase-contrast inverted microscopy.
Transwell migration assay. 2.5 × 10 4 HUVECs were incubated in 500 μl serum-free MEM-alpha medium and added to the upper chamber in a Transwell 24-well plates (Corning, USA). The lower chamber was precoated with PF127 with or without SAP, and 750 μl complete medium with or without HUCMSCs-CM was placed in the lower chamber. After 12 h of incubation, the lower surface of HUVECs were stained with crystal violet for 15 min. Finally, the migrated HUVECs were photographed and counted with a microscope.
In vitro antimicrobial activity assay. 100  Skin wound model establishment. The full-thickness skin wound modeling in mice was conducted as previously reported 12 . In brief, the mice were anesthetized and placed prone on the operating table. After successively shaving off the hair in the back area with electric scissors and depilatory wax, a full-thickness round wound with a diameter of 12 mm was made with a biopsy punch on the back of each mouse. All the mice were blindly randomized into four groups (n = 6): (1)  Statistical analysis. The statistical tests were carried out via GraphPad Prism 6.0. The differences between two or more groups were compared via the one-way ANOVA followed by Tukey's posttest. P less than 0.05 was considered statistically significant.  Fig. 1A (Fig. 1B). The morphological features of hydrogels were observed by SEM after lyophilization (Fig. 1C). Both PF127 hydrogel and PF-127/ HUCMSCs/SAP hydrogel presented interconnected and similar porous microstructures. The loose and porous network structure exhibits the good absorptivity and breathability of PF-127/HUCMSCs/SAP hydrogel, which facilitates skin wound healing.

SAP improves the survival and migration of HUCMSCs encapsulated in PF-127 hydrogel.
To determine the biological role of SAP on HUCMSCs encapsulated in PF-127 hydrogel. HUCMSCs were cocultured in PF-127 hydrogel with or without SAP for 24 h, and cell viability was determined by CCK-8 assay. As shown in Fig. 2A, the cell viability was markedly reduced after HUCMSCs were encapsulated in PF-127 hydrogel. The co-treatment with SAP (400 μM) could remarkably counterbalance the suppressive effect of PF-127 on the cell viability of HUCMSCs. We further explored whether apoptosis contributed to the HUCMSCs death caused by PF-127 ( Fig. 2B). In accordance with the results of the CCK-8 assay, results of the DAPI staining assay showed the percentage of DAPI-positive PF-127-encapsulated HUCMSCs was significantly higher than that of the control cells. It is noted that the promoting effect of PF-127 on the apoptosis of HUCMSCs was attenuated by the supplementation of SAP (400 μM). Whereas there was no statistical difference in the regulation of cell viability and apoptosis as the concentration of SAP was raised to 800 μM. These discoveries showed that the addition of SAP markedly improves HUCMSCs viability but attenuates the apoptosis of HUCMSCs in PF-127 encapsulation.
We then explored whether PF-127 combined SAP promoted the migration of HUCMSCs by cell wound scratch assay. As anticipated, the migration of HUCMSCs was markedly inhibited by PF-127 (Fig. 2C). Meanwhile, we found that the migration of HUCMSCs was improved significantly following 400 μM SAP or 800 μM SAP applications as compared with the PF-127 group. Fig. 3, PF-127 exerts almost no inhibitory effect on the growth of E. coli and S. aureus. The PF-127/SAP hydrogel exerted a strong bacteriostatic function; the colony number in PF-127/SAP (800μM) group was the least abundant compared with other groups. Collectively, the combination of PF-127 and SAP demonstrated powerful antibacterial properties against both Gram-positive and Gram-negative bacteria. Thus, these findings suggest that PF-127/ SAP hydrogel could improve HUCMSCs survival as well as reduce the risk of wound infection.

PF-127 plus SAP combination contributes to HUCMSCs-mediated angiogenesis in vitro.
Subsequently, tube formation and transwell migration assays were employed to investigate the biological characteristics of HUVECs cultured with or without HUCMSCs-CM in different encapsulation conditions. The tube formation and migration of HUVECs were not obviously Influenced after incubating with 20% PF-127 solution (Fig. 4A,B). HUVECs cultured in PF-127/HUCMSCs-CM formed more tubules per field of view (FOV) than Similarly, the transwell migration assay showed that compared with the PF-127 group, PF-127/HUCMSCs-CM significantly promoted the number of migrated HUVECs, and the addition of SAP further enhanced the PF-127/HUCMSCs-CM-induced HUVECs migration (Fig. 4D). These findings showed that SAP could further enhance the restoration of HUVECs function induced by HUCMSCs-CM.

PF-127 plus SAP combination facilitates HUCMSCs-mediated skin wound healing in vivo.
The above experiments have confirmed that PF-127 coupled with SAP facilitates HUCMSCs-mediated wound closure in vitro. Further study was done to explore the hypothesis that PF-127 plus SAP could contribute to HUC-MSCs-mediated wound healing using the full-thickness skin wound model. Similar to our prediction, compared with the control group, remarkable accelerating effects of skin wound recovery have been observed in the PF-127 hydrogel group. The application of PF-127/HUCMSCs led to a further decline in residual wound area at day 9 post-surgery compared to the PF-127 group (Fig. 5). The wound in the PF-127/HUCMSCs/SAP group healed better on day 9 after transplantation than in the other groups. At 15 days after surgery, the skin wounds in the PF-127/HUCMSCs/SAP group almost entirely healed, while other groups still had varying degrees of visible unhealed wounds.
PF-127 coupled with SAP promotes HUCMSCs-mediated dermis regeneration and collagen deposition. Histological analysis was conducted to explore further the effect of the PF-127/HUCMSCs/ SAP hydrogel on wound healing. As seen in Fig. 6A, at 15 days after surgery, the inflammatory response can be observed in all four groups, while the most serious inflammatory response was seen in the PBS group, which is characterized by local hemorrhagic focus, many inflammatory cells but few collagen fibers, a substantial decline in connective tissue. In the group of PF-127/HUCMSCs, the inflammation was lessened or subsided. In sharp contrast, tissues in the PF-127/HUCMSCs/SAP group showed the least inflammatory cells. The dermis thickness in the PF-127/HUCMSCs/SAP group was thicker than other groups (Fig. 6B), along with a relatively intact epi- www.nature.com/scientificreports/ thelium layer and abundant fibroblasts in the dermis. H&E staining also illustrated that the PF-127/HUCMSCs/ SAP group exhibited a smaller residual wound area than other groups at day 15 post-surgery. The statistical data also verified that the quantity of newborn hair follicles was markedly higher in all scaffold groups when compared with that in the control group (Fig. 6C), and the PF-127/HUCMSCs/SAP group still showed the highest amount of hair follicles. Additionally, the collagen fibers disposition in the wound area tissues was determined by Masson staining. The present study showed that the proportion of collagen fibers deposited at the regenerated dermis area in the PF-127/HUCMSCs/SAP group is much higher than in other groups (Fig. 7).

PF-127 combined with SAP improves HUCMSCs-mediated cell proliferation and angiogenesis in vivo.
We further explore the mechanism underlying PF-127 plus SAP combination improves HUCM-SCs-mediated wound healing. Ki-67, CD31 and, VEGF, which are related to proliferation and angiogenesis, are evaluated by immunohistochemical staining. As shown in Fig. 8, the Ki-67 positive cells in the tissues of the PF-127/HUCMSCs/SAP group were significantly higher compared to other groups. Our study showed that the number of newly formed blood vessels in the PF-127/HUCMSCs/SAP group was significantly increased than the other groups. Furthermore, the expression of VEGF was conducted to evaluate angiogenesis. VEGF-positive cells could be found in all four groups, while its positive cell number was highest in the PF-127/HUCMSCs/ SAP group. Therefore, these results revealed that PF-127 plus SAP combination improves HUCMSCs-mediated wound healing by promoting cellular proliferation and angiogenesis.

Discussion
An ideal wound dressing material is not restricted to preventing external infections, but also to guiding skin cell infiltration. Adequate vapor permeability, easily removable, and good flexibility are all essential requirements for wound dressing 26,27 . Among these various wound dressings, polymeric hydrogels offer many advantages, such as good hydrophilicity, excellent biocompatibility, and adjustable physical properties, which are identified as one of the most promising wound dressing material candidates 13 . As one of the most commonly used polymeric hydrogels, the porous structure of PF-127 acts as a solid barrier against external infections, provides moisturization and absorbs extrudates 14 . In the present study, the local application of PF-127 hydrogel alone www.nature.com/scientificreports/ could promote skin wound healing, but the effect could be more satisfactory, similar to previous reports 15,18,19 .
Recently, a tissue engineering-based strategy has shown that the application of biomaterial-based scaffolds coupled with stem cells, drugs, or biologically active factors in the regeneration of skin wounds has gained more popularity and attention [10][11][12][13] . In the present study, 20% PF-127 served as a delivery system to load HUCMSCs/ SAP complexes, and the therapeutic effect of the PF-127/HUCMSCs/SAP hydrogel on full-thickness cutaneous wound healing was investigated. Many basic and clinical studies have proven that HUCMSCs are a promising drug candidate for wound healing in human and animal skin defects due to their beneficial properties, such as the stimulation of angiogenesis, the regulation of immunity, and the promotion of cell regeneration [7][8][9] . However, due to stem cell suspension run-off after topical injection at the transplanted site, less than 1% of transplanted stem cells remain at the injection site for more than a week. So, the therapeutic efficacy of stem cells was markedly attenuated by poor cell engraftment. In order to solve this issue, embedding cells in biomaterial-based scaffolds to confine injected cells at the location of transplantation without run-off has been deeply assessed [15][16][17] . IVFK hydrogels provide cell attachment sites for mouse myoblasts, significantly prolonging cell survival and supporting their expansion and differentiation into myotube myocytes 28 . It was reported that the combination of engineered fibrin hydrogel and MSCs could localize more cells at the wound site, which revealed better wound repair effects than the transplantation of MSCs alone 29 . Another study demonstrated that stem cells encapsulated in chitosan hydrogel sustained a high cell density for a long time, and this combination significantly promoted wound healing 30 . The thermo-sensitive feature of PF-127 hydrogel facilitates cell attachment and retention at the transplantation location, which prompted PF-127 to be widely used in the field of tissue regeneration 14 . In our study, PF-127/ HUCMSCs hydrogel could be adequately filled in irregular skin wounds evenly in a solution condition. After transplanting onto the skin defects, the fluidity of PF-127/HUCMSCs hydrogel gradually decreased with the temperature increase. The hydrogel becomes condensed eventually and separates the skin defect from the external environment to reduce the possibility of wound infection. The application of PF-127/HUCMSCs remarkably speeds up the repair of skin wounds compared to the PF-127 group. At the same time, PF-127 can upregulate the www.nature.com/scientificreports/ expression level of intracellular ROS, resulting in cytotoxic effects on encapsulated cells 15,18,19 . In line with these findings, results from the current study consistently uncovered that the survival and migration of encapsulated HUCMSCs were significantly attenuated after incubation in 20% PF-127 hydrogel for 24 h. Therefore, poor cell survival and biological characteristics of MSCs in PF-127 encapsulation were other obstacles that limited the therapeutic efficacy of stem cells.
Researchers have made a lot of attempts to improve the therapeutic effect of transplanted stem cells. A recent study has shown that red OLED light can enhance the angiogenesis, cell adhesion, and migration capabilities of human adipose-derived mesenchymal stem cells 31 . Many studies have focused on the addition of cell membrane-stabilizing agents to the PF-127 hydrogel to improve cell survival. Hydrocortisone was generally added to the PF-127 gel to improve cell viability, such as HepG2 and HMEC-1 cells 32 . However, hydrocortisone has an immunosuppressive effect on the body, which adversely affecting wound recovery. Meanwhile, the application of natural product vitamin C can increase the viability of PF-127-encapsulated BMSCs 32 . Vitamin C has a powerful antioxidant effect, and vitamin C alone can effectively inhibit bacterial growth and promote wound recovery, so it is an ideal cell membrane stabilizer 33 . As one kind of vitamin C analog, SAP is characterized by higher physical-chemical stability than ascorbic acid 21 . This study also observed that SAP can effectively inhibit bacterial growth in vitro. Furthermore, supplementation of SAP attenuates the cytotoxic effect of PF-127 and enhances cell survival in rat MSCs encapsulation 15 . Given that SAP has a strong ROS scavenging ability, we hypothesize that adding SAP to PF-127 can improve the survival of embedded HUCMSCs, thereby increasing the repair effect of HUCMSCs on skin wounds. Our results confirmed this conjecture. In the present study, CCK-8 and apoptosis assays demonstrated that 400 μM SAP greatly promoted the survival of HUCMSCs encapsulated in PF-127. Our in vivo data revealed that PF-127 plus 400 μM SAP significantly contributed to HUCMSCs-mediated fullthickness wound healing, dermis regeneration, and cell proliferation in mice. These in vivo findings parallel the alterations found in vitro. The current study reveals for the first time that PF-127 and SAP composite improve the efficacy of HUCMSCs-mediated wound healing both in vitro and in vivo. We propose that PF-127 enhances the in-situ residence time of HUCMSCs as well as the antioxidative effect of SAP, which contributes to the great wound healing effect of PF-127/HUCMSCs/SAP hydrogel, while the underlying mechanism remains unclear.   www.nature.com/scientificreports/ The formation of new blood vessels plays a vital role in orchestrating wound healing. The non-healing nature of chronic wounds is primarily attributable to impaired angiogenesis, which fails to supply adequate nutrients to the injured site 34 . Several lines of evidence suggest that stimulation of angiogenesis is an essential mechanism by which MSCs accelerate wound recovery [34][35][36] . The proliferation and migration of endothelial cells are considered to be two critical steps in angiogenesis 34 . In vitro experiments, we found that the tube formation and migration of HUVECs were not significantly influenced by PF-127, which demonstrated the biocompatibility of PF-127 hydrogel. Recent studies have shown that stem cells can release a variety of bioactive molecules, such as growth factors, cytokines, chemokines, extracellular matrix (ECM), and small molecules. These bioactive molecules are present in the CM or secretomes, and stem cells use these bioactive molecules to help tissue regeneration 37 . Our results showed that HUCMSC-CM could promote angiogenesis and migration of HUVECs in vitro, suggesting that HUCMSCs may promote the functional recovery of vascular endothelial cells through paracrine effects. This acellular approach has enormous advantages over traditional treatment options and is therefore receiving increasing attention. Furthermore, the pro-angiogenic effect of HUCMSCs is further reinforced by the application of SAP in vitro and in vivo; these results are similar to previous literature reports 38 . Not yet known is the signaling mechanism by which HUCMSCs promote angiogenesis.
VEGF is a definite factor that promotes the permeability of capillaries, the mitogenesis and migration of vascular endothelial cells, matrix degeneration, and vessel tube formation 39 . VEGF alone or in combination with other therapy has been employed to treat chronic wounds 40 . Our study showed that the protein level of VEGF, which almost had no expression in the wounds of PF-127 group, was markedly increased in PF-127/HUCMSCs and PF-127/HUCMSCs/SAP groups, which is coincident with the immunostaining staining results of CD31. Taken together, PF-127/HUCMSCs/SAP hydrogel can efficiently promote the angiogenesis of traumatic skin Figure 8. Light microscopic pictures of immunohistochemical staining of Ki-67, CD31, and VEGF in the wound bed and surrounding normal tissues. *P < 0.05 versus PBS group. ## P < 0.01, ### P < 0.001 versus PF-127 group. ▲ P < 0.05, ▲▲▲ P < 0.001 versus PF-127/HUCMSCs group. n = 6 independent samples. Data are presented as mean ± SD. Error bars represent SD. Scale bar 100 μm. www.nature.com/scientificreports/ wounds, and the promoted VEGF level plays a vital role in accelerating angiogenesis, thereby promoting tissue repair.
Taken together, the present study discloses a novel and effective hydrogel scaffold to deliver HUCMSCs to treat the traumatic cutaneous defect. SAP is identified as an effective cell membrane stabilizing agent that dramatically improves the survival of PF-127 encapsulated HUCMSCs via promoting cell proliferation, antibacterial action, and neovascularization, and the up-regulation of VEGF may play a crucial role in PF-127/HUCMSCs/ SAP induced wound healing. Our results showed that the PF-127/HUCMSCs/SAP hydrogel showed great biological activity and antibacterial properties and has excellent potential in accelerating wound healing, especially in infected wounds.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.